Stimulated emission depletion microscopy with a single depletion laser using five fluorochromes and fluorescence lifetime phasor separation

Abstract

Stimulated emission depletion (STED) microscopy achieves super-resolution by exciting a diffraction-limited volume and then suppressing fluorescence in its outer parts by depletion. Multiple depletion lasers may introduce misalignment and bleaching. Hence, a single depletion wavelength is preferable for multi-color analyses. However, this limits the number of usable spectral channels. Using cultured cells, common staining protocols, and commercially available fluorochromes and microscopes we exploit that the number of fluorochromes in STED or confocal microscopy can be increased by phasor based fluorescence lifetime separation of two dyes with similar emission spectra but different fluorescent lifetimes. In our multi-color FLIM-STED approach two fluorochromes in the near red (exc. 594 nm, em. 600–630) and two in the far red channel (633/641–680), supplemented by a single further redshifted fluorochrome (670/701–750) were all depleted with a single laser at 775 nm thus avoiding potential alignment issues. Generally, this approach doubles the number of fully distinguishable colors in laser scanning microscopy. We provide evidence that eight color FLIM-STED with a single depletion laser would be possible if suitable fluorochromes were identified and we confirm that a fluorochrome may have different lifetimes depending on the molecules to which it is coupled.

Combining sophisticated fast FLIM, confocal microscopy, and STED nanoscopy for live-cell imaging of tunneling nanotubes

“…In addition, lifetime denoising, lifetime imaging, and lifetime dye unmixing could be properly used or combined to improve the signal-to-noise ratio, to identify the different lifetimes, or to separate the dye signals in order to reveal a TNT in living H28 cells ( Figs 2 and S5 ). Because a STED depletion laser altered the fluorescence lifetime characteristics ( Gonzalez Pisfil et al, 2022 ) ( Fig S5A ), the achievement of multi-labeling experiments with spectrally close dyes became more difficult and only a strong enhancement may reveal specific signals, for example, actin ( Fig S5B ). Therefore, a dedicated “HyD detector–phasor plot circle” for each dye was necessary to perform accurate post-processing.…”

“…When two detectors were needed in double-labeling experiments through STED nanoscopy, a second detector HyD-S was selected because it displayed a rather good sensitivity all over the spectrum ( Schweikhard et al, 2020 ; Bénard et al, 2021 ). The new generation of HyD detectors, combined with fast electronics, also offered new perspectives for multiplexing and lifetime acquisition in living cells including user-friendly solutions, and FLIM became consequently more accessible to cell biologists ( Alvarez et al, 2019 ; Bitton et al, 2021 ; Gonzalez Pisfil et al, 2022 ). In order to bypass the spectral dye overlapping during multiplexing, the fluorescence lifetime separation was an alternative to the limited fluorescence spectral separation.…”

Spatial resolution enhancement in photon-starved STED imaging using deep learning-based fluorescence lifetime analysis

“…Our next task is to demonstrate STED-flimGANE in imaging organelles in live cells. As one of the key advantages of phasor analysis is the differentiation of multiple fluorophores with different lifetimes excited by a single excitation source, 44,45 with recent development in fluorescence lifetime tuning strategies in fluorophores and protein tags, [46][47][48][49] we envision that our STED-flimGANE can also be adapted for multiplexed STED imaging.…”

Protein nanobarcodes enable single-step multiplexed fluorescence imaging

“…A number of other strategies have also emerged, which could be employed for multichannel observations. First, one could rely fluorescence lifetime detection, to separate spectrally similar fluorophores [ 38 ], or, in a more advanced implementation, one could use single-molecule spectroscopy, for the same purpose [ 39 ]. An often used approach for multiplexing, as mentioned in the introduction, is DNA-PAINT, for which all our nanobodies are readily available, some have already been used for PAINT multiplexing [ 4 ].…”